Construction of Recombinant Human Soluble TIM-3 Cell Line and Expression of Its Secretory Protein

This study constructed a stable transfected cell line carrying the target gene of TIM-3 (Fc) by lentivirus infection and detected the expression of TIM-3 (Fc) secretory protein. Specific primers were designed for PCR amplification of TIM-3 (Fc) fragment, and positive clones were identified in recombinant lentivirus expression vector pCD513. Plasmid pCD513-TIM-3 (Fc), pSPAX2 and pMD2.G were co-transfected into 293T cells to package lentivirus, and lentivirus was used to infect suspension HEK293 cells. Finally, stable transfected cell line 293/Tim-3 was obtained through puromycin screening. qRT-PCR (quantitative real-time PCR) and WB (Western blot) were used to detect the expression of TIM-3 (Fc) target gene at the levels of transcription and translation, and TRFIA was used to detect the ability of 293/TIM-3 cells to produce TIM-3 (Fc) secretory protein. pCD513-TIM-3 (Fc) lentivirus expression vector was successfully constructed, and the detection results of qRT-PCR, WB and TRFIA indicated that the cell line 293/TIM-3 successfully expressed TIM-3 (Fc) secretory protein. The recombinant human soluble TIM-3 stable transfected cell line 293/TIM-3 is constructed and TIM-3 (Fc) secretory protein expressed, which provides the foundation for further study on the biological function of soluble TIM-3 protein.